Microbiological oxidation of alkylbenzenes



United States Patent 3,383,289 MICROBIOLOGICAL OXIDATION OFALKYLBENZENES Richard L. Raymond, Wilmington, Del., and Virginia W.

Jamison, Prospect Park, Pa., assignors to Sun Oil Company, Philadelphia,Pa., a corporation of New Jersey No Drawing. Filed Nov. 24, 1965, Ser.No. 509,621 20 Claims. (Cl. 195-28) ABSTRACT OF THE DISCLOSURE C7-C1oorganic acids which are methyl-substituted muconic acids and/or2,3-dihydroxybenzoic acids are prepared by microbiological oxidation ofC -C methylbenzenes having 1-4 methyl groups and at least twoconsecutive ring carbon atoms by the action of orthodihydroxylating andnon-decarboxylating strains of Nocardia.

This invention relates to the fermentation of methylsubstituted benzenehydrocarbons under conditions resulting in the production of either orboth of two types of organic acids. More specifically the inventionpertains to the microbiological oxidation of methylbenzenes having 7-10carbon atoms per molecule by means of specifically acting strains ofmicroorganisms of the genus Nocardia. The strains used in accordancewith the invention are characterized by their ability to produce from CC methylbenzenes either a methyl-substituted muconic acid or adihydroxybenzoic acid or both as hereinafter described.

The microbiological oxidation of aromatic hydrocarbons by means ofvarious types of microorganisms has been considered heretofore byseveral investigators. Numerous prior art reports dealing with thissubject matter have been published. Davis and Raymond in an articleappearing in Applied Microbiology, vol. 9, No. 5, September 1961, pages383388, refer to various prior art publications on this subject. Theseauthors therein and also in their United States Patent No. 3,057,784have described the oxidation of alkylbenzenes by means of certaincultures of Nocardia. In all cases oxidation occurred only on the alkylgroup, so that acidic products such as benzoic acid, phenyl acetic acidand phenyl acrylic acid were obtained depending upon the alkylbenzeneselected. When the alkyl substituent had an odd number of carbon atoms,the principal oxidation product generally was benzoic acid, while for aneven number of carbon atoms it was phenyl acetic acid. The resultsreported gave no indication that any strains of Nocardia could effect adirect hydroxylation of the benzene ring.

A recent review of microbiological oxidations of hydrocarbons, includingalkylbenzenes, appears in the textbook Advances in Enzymology, vol. 27,pages 469-546 (Interscience Publishers, 1965). On page 496 the reviewauthors point out that heretofore in microbiological oxidation ofalkylbenzenes generally, most if not all microorganisms do not initiallyattack the phenyl ring but rather attack an alkyl substituent. In oneinstance it is reported that toluene was converted to catechol(1,2-dihydroxybenzene) by Pseudo'monas aeruginosa but the pathwayinvolved initial oxidation of the methyl group leading to the formationof benzoic acid, followed by decarboxylation of the carboxyl radical and1,2-dihydroxylation. No case is reported in which dihydroxylation of thephenyl ring of an alkylbenzene occurred without prior formation of thecarboxyl radical from the alkyl substituent followed by decarboxylationthereof. Only in the case of benzene itself has it been found that somemicroorganisms are capable of effecting direct dihydroxylation of thering.

In those cases where benzene itself has been oxidized microbiologically(see pages 506-510 of the aforesaid textbook) catechol is formed. It hasbeen found that the metabolic mechanisms of a few microorganisms arecapable of causing further oxidation of the catechol while also bringingabout ring splitting between the adjacent hydroxyl groups. This hasresulted in the formation of muconic acid usually in the form of thecis,cis-isorner. The overall conversion can be represented as follows:

0 O O H benzene catechol ois,cis-muconic acid While a few microorganismshave been shown to effect the conversion of benzene in this manner, noanalogous conversion of an alkylbenzene appears to have been disclosed.Hence the preparation from an alkylbenzene of al-carboxy-2,3-dihydroxybenzene having the same number of carbon atoms asthe parent hydrocarbon or of an alkylsubstituted muconic acid appears tohave been unknown heretofore.

We have now discovered that there are strains of microorganisms of thegenus Nocardia which can in a novel manner oxidize methyl-substitutedbenzenes that have at least two consecutive unsubstituted ring carbonatoms. These hydrocarbon substrates used in the present invention arethe C7-C10 methyl-substituted benzenes having 14 methyl groups and atleast two adjacent ring carbon atoms containing no substituents. In onenovel aspect a methyl group attached to a carbon atom next to the twounsubstituted carbons will convert to a carboxyl group while alsodiorthohydroxylation of the ring at the two adjacent open carbon atomswill occur. Further this will take place without decarboxylationoccurring. The result of these metabolic reactions is the formation of2,3-dihydroxybenzoic acid or homologues thereof depending upon thestarting methylbenzene used. For example, from pxylene suitable strainsof Nocardia can form 2,3-dihydroxy-p-toluic acid (referred to 'forconvenience herein as DHPT). In such cases it appears that p-toluic acid(referred to herein as PTA) is a precursor in the metabolic sequence toDHPT and hence substantial amounts of PTA generally are accumulatedwhenever DHPT is produced. This is particularly true in later stages ofthe fermentation. Analogous accumulations of other non-hydroxylatedbenzoic acids also usually take place concurrently with the productionof other 2,3-dihydroxybenzoic acids in this embodiment of the invention.

We have further discovered that some strains of Nocardia exhibit stillanother novel feature of oxidation for C -C methylbenzenes having atleast two consecutive unsubstituted ring carbon atoms. These strains arecapable of diorthohydroxylating the ring without any attendant oxidationof the methyl substituent and then causing ring cission between thehydroxylated carbon atoms. This ring splitting is the result of furtheroxidation which converts the hydroxylated carbons to carboxyl groups.Thus dimethyl-muconic acid (referred to herein for convenience as DMMA)can be produced by biological oxidation as illustrated by the following:

6 f V f m i H l o o o H O O O H O H I l O C O p-xylene3,6-dimethyla,a-dimethylcatechol muconic acid (DMMA) The substitutedcatechol shown in the equation is a transitory intermediate in themicrobiological reaction and generally does not appear in large amountsin the fermentation broth, although in some cases small amounts mayaccumulate and be present in the final product. As indicated, the DMMAobtained from the fermentation of p-xylene is in the form of itscis,cis-isomer. This isomer can readily be isomerized to thecis,trans-isomer and/ or the trans,trans-isomer under appropriateisomerization conditions.

It has been found that from C- -C methylbenzenes suitable strains ofNocardia will produce either 2,3-dihy droxybenzoic acids or a homologueof muconic acid or both. Either type acid will have the same number ofcarbon atoms as the parent hydrocarbon. The muconic acid homologue willalso have the same number of methyl substituents as the parenthydrocarbon while the dihydroxybenzoic acid will have one less methylgroup. By way of example, from p-xylene some strains of Nocardia willproduce DHPT but no DMMA while some will produce both DHPT and DMMA.Among the latter strains some can be made to produce DMMA to thesubstantial exclusion of DHPT under selected fermentation conditions. Aspreviously stated, whenever DHPT is produced, a substantial amount ofPTA generally is also formed.

Suitable types of Nocardia for practicing the present invention areherein referred to as orthodihydroxylating and non-decarboxylatingstrains. By the term orthodihydroxylating is herein meant that themicroorganism is capable of forming on the ring of the methylbenzene twohydroxyl groups which are ortho to each other and one of which is orthoto a substituted carbon atom of the benzene ring. This term, of course,does not indicate that the product ultimately formed in the fermentationnecessarily contains any hydroxyl groups, as the muconic acid homologuesin fact do not. The term non-decarboxylating" signifies that themicroorganism does not cause destruction of carboxyl groups, which havebeen formed during the oxidation, by releasing carbon dioxide therefrom.Thus it is characteristic of fermentations practiced according to thepresent invention that any carboxyl group formed remains intactthroughout the fermentation.

Strains of Nocardia which are orthodihydroxylating andnon-decarboxylating have been found among various species occurring innature, including species classified in accordance with Bergeys Manualas Nocardia corallz'na, Nocardia salmonicolor and Nocardia minima. Forthe present purpose appropriate strains of Nocardia corallina generallyare preferred. Numerous attempts have been made to find among otherknown hydrocarbon-consuming genera strains which have similarorthodihydroxylating and non-decarboxylating characteristics. Among thegenera tried are species of Brevibacterium, Pseudomonas, Streptomyces,Candida and Bacillus. Thus far, however, none of these has shown thedesired characteristics that are exhibited by suitable strains ofNocardia used in practicing the present invention.

In accordance with the invention a methylbenzene of the C -C range isconverted to an organic acid product by means of a Nocardiamicroorganism having the abovedescribed properties. The startinghydrocarbon can be any mono-, di-, trior tetramethylbenzene which has atleast two consecutive unsubstituted ring carbon atoms. More particularlythe following methylbenzenes can be used: toluene; 0-, mor p-xylene;pseudocumene; hemirnellitene; and prehnitene. The product acid is eithera methyl-substituted higher homologue of muconic acid or a2,3-dihydroxybenzoic acid or both. When a 2,3-dihydroxybenzoic acid isproduced, it is generally associated with the correspondingnon-hydroxylated benzoic acid which, as previously indicated, seems tobe the metabolic precursor for the dihydroxylated product. Theconversion is effected by subjecting the methyl-substituted benzene inthe presence of a nutrient medium and under fermentation conditions tothe action of an orthodihydroxylating and non-decarboxylating strain ofNocardia. After the desired fermentation oxidation has occurred, atleast one of the acids of the foregoing types is recovered from thefermentation broth. For some of the microorganism strains both a muconicacid homologue and a 2,3-dihydroxybenzoic acid are produced andrecovered, whereas in other cases substantially only one or the other ofthe product acid types is made.

The following table shows specifically the hydrocarbon substrates whichcan be used in practicing the invention and the acid products obtainabletherefrom. The table lists the substrates and product acids both by nameand by formula.

OBTAINABLE PRODUCT ACIDS Hydrocarbon Aromatic Acid Substituted MuconicAcid Substrate (I) (I) OH 2 r OH HOOOC=C-C=C-COOH I I C G (p-xylene)(2,3-dihydroxy- (a,a-dimcthyl muconic acid) p-toluic acid) (I) (I O OH OO OH r 6 OH HOOC-C=OJJ=C-COOH (Dseudocumcne) (2,3-dihydroxy-4,6-di-(u,a,5-trimethyl muconic acid) methyl benzoic acid) O- OH(2,3-dihydroxy4,5-dimethyl muconic acid) (I) C O OH C- C OH I i F 1 C- OOH HOOCC=CC=CC OOH (hemimellitene) (2,3-dihydroxy-5,6-di- (01,8,5'trimethyl muconic acid) methyl-benzoic acid) 0 C O OH 0 C OH I l 0 0 OHHOOC-C=CC=C-COOH (prchnitene) (2,3-dihydroxy-4,5,6-(a,a,/3,B-tetramethyl muconic acid) trimethyl-benzoic acid) From thetable it can be seen that the dihydroxylating and non-decarboxylatingstrains of Nocardia used in practicing the invention can convert each ofthe methylbenzenes listed to an aromatic acid which is a2,3-dihydroxybenzoic acid and/or to a muconic acid homologue which hasone methyl substituent at the alpha position and which may or may nothave other methyl groups depending upon which hydrocarbon substrate isused. For all substrates except pseudocumene only one dihydroxylatedaromatic acid and/or one muconic acid homologue results from themetabolic reactions shown by these Nocardia strains. In the case ofpseudocumene there are two dihydroxylated aromatic acid isomers whichmay appear, which isomers differ only in the position of one methylgroup. It can be seen that all of the acid products have the same numberof carbon atoms as the starting hydrocarbon, that the muconic acid typeproduct has the same number of methyl substituents as the substrate andthat the aromatic type acid has one less methyl substituent, the latterhaving been converted to a carboxyl group.

Specific microorganisms which have been used for the present purposeinclude the following:

(1) A wild-type strain obtained from soil in Alabama, havingcharacteristics approximating those set forth for Nocardia coraltina inBergeys Manual and hence classified as such species. A culture of thisstrain has been deposited with the American Type Culture Collection inWashington, D.C., under the number ATCC 19,070. Colonies of thismicroorganism have an orange color.

(2) A reddish colored mutant obtained by ultraviolet irradiation of ATCCNo. 19,070. The mutant has also been deposited with the American TypeCulture Collection and has been designated as ATCC No. 19,071.

(3) A strain isolated from Pennsylvania soil and likewise classified asNocardia corallina. This microorganism is orange colored like thefirst-mentioned wild-type specimen but shows distinct differences inenzymatic oxidative characteristics as hereinafter described. A culturedeposit of this strain has been designated as ATCC No. 19,148.

(4) A soil isolate having characteristics approximating those given inBergeys Manual as Nocardia salmonicolor and hence so classified. Aculture deposit has been identified as ATCC No. 19,149.

(5) Another soil isolate classified as per Bergeys Manual as Nocardiaminima and designated as ATCC No. 19,150.

Conc., g./l. of H 0 2 MgSO -7H O Na CO 0.1 CaCl -2H O 0.01 MnSO -H O0.02 FeSO 7H O 0.005 Na KPO 3.0 KH PO 2.0 Urea 2.0

This mineral salt composition normally would have a pH of about 7.1.When it is desired to carry out the fermentation at a pH below 7, as isthe case when the object is to maximize the production of the muconicacid homologue, the amount of KH PO relative to Na HPO can be increasedto reduce the pH to a lower level.

The process of the invention is generally carried out at a temperaturewithin the range of -40 C. and preferably at 28-32" C. under aerobicconditions with agitation. The nutrient medium should have a pH in therange of 4 to 9 and more desirably 6-8. When the Nocardia strain in onecapable of effecting ring splitting to form a muconic acid homologue,production of such acid can be maximized by maintaining the pH in therange of 6-7, with a pH of about 6.8-7.0 usually being best. When adihydroxybenzoic acid is the preferred product, its formation can befavored by operating at a pH in the range of 7-8 and a level of about7.8 is generally preferred.

In preparing a Nocardia culture for use in the present process, a sampleof a suitable Nocardia strain from a slant is transferred to a shakeflask containing mineral salts solution and a suitable carbon source forgrowth. The carbon source can be a suitable hydrocarbon such ashexadecane. saturates derived from kerosene or toluene, or acarbohydrate or hydrolyzed protein. Preferably the carbon sourcematerial is added periodically in small amounts during incubation. Insome cases it may be desirable also to have growth-stimulating materialssuch as peptone, beef extract or yeast extract present, although this isoften not necessary. In the case of the mutant ATCC No. 19,071 referredto above, such material should be supplied since this oragnism, unlikethe parent wildtype ATCC No. 19,070, requires a source of the vitamin,p-aminobenzoic acid, at least for initial growth and such material canprovide this growth factor. The mixture is incubated at 30 C. andhexadecane (or other carbon source material) is added from time to timeas the cell growth takes place, preferably being added in increasingamounts. After an incubation period typically of 24 hours, the cells canthen be used for purpose of the invention.

The fermentation can be carried out by subjecting the methylbenzenesubstrate in the presence of the nutrient medium to action of theNocardia organism under either growth or non-growth conditions. Whengrowth conditions are employed, a sample of the inoculum prepared asabove described is added to a mineral salts medium in a fermentor andthe cells are first grown at 30 C. on hexadecane, for example, for about24 hours without any addition of the methylbenzene substrate. After goodgrowth has been obtained, periodic additions of the methylbenzene, alongwith additional amounts of hexadecane to sustain growth, are made andthe fermentation is continued until maximum yield of the desireddihydroxybenzoic acid and/or muconic acid homologue is obtained. A totalfermentation time of 96 hours usually is typical for obtaining maximumproduct yield.

When the Nocardia organism is used under non-growth conditions forpracticing the invention, cells grown as previously described areseparated from the broth by centrifuging and washed with phosphatebuffer solution and then are resuspended in phosphate buffer solution.The suspension is maintained at say 30 C. and the methylbenzenesubstrate is added periodically in incremental amounts or continuouslywhile the mixture is being aerated and stirred. Addition of thesubstrate is continued until the fermentation has given an optimum yieldof the desired acid product.

After the fermentation has been completed, the cells are separated fromthe broth by centrifugation and the clear broth can then be processed inany suitable manner for recovery of the acid products. In cases where amuconic acid homologue (e.g., DMMA) has been produced it can beseparately recovered by acidifying the broth with a mineral acid (e.g.,HCl) to a pH of say 2, whereupon the DMMA will selectively precipitatefrom solution and can be separated by filtration and then purified bywater washing. Any DHPT and PTA present in the broth will remain in theaqueous solution and can be removed therefrom by extraction with asuitable solvent such as ether, dioxane or amylacetate. The DI-IPT andPTA can thereafter be separated from each other chromatographicallyemploying an anion exchange resin. As an alternative procedure, theacidified aqueous solution obtained upon filtering out the precipitatedDMMA can be evaporated to obtain a concentrate of DHPT and PTA and theseproducts can then be separated from each other by extraction of theconcentrate with a suitable selective solvent.

A preferred microorganism for practicing the invention to produce themuconic acid type of derivative is Nocardia corallina ATCC No. 19,070mentioned above. Cultural and physiological characteristics whichidentify and distinguish this microorganism are as follows.

Staining characteristics:

Age24 to 168 hours Gram-gm+, granules Cell morphology:

Form-rod with branching in young culture (0-48 hrs.) Motilitynon-rnotileSize 24 hours1l.5 by 3-20 microns, branching 48 hours-0.5-1.5 by 1-5microns, some branch ing 72 hours-1 by 1-2 microns Agar colonies:

Age-72 hours Form-Circular Elevationconvex Surface-butyrous MarginentireChromogenesis-orange Agar stroke:

Age-72 hours Form-filamentous Consistency-butyrous Chromogenesis-lightorange Nutrient broth:

Surface growthflocculent Subsurface growthnone Amountfair growthSediment-granular, orange Gelatin stab:

Liquefaction-none Growth-none Potato dextrose agar:

Age72 hours Growthnone Potato slant:

Age-72 hours Chromogenesis-deep orange Consistency-butyrous Glucoseagar:

Age-72 hours Chromogenesis-light orange, cream Consistency-butyrousAction on sugars:

'Maltose-no acid, no gas, very good growth Sorbitolno acid, no gas, verygood growth Dextrose-no acid, no gas, growth Mannito1no acid, no gas,slight growth Lactose-slight alkaline, no gas, good growthL-arabinose-slight alkaline, no gas, growth Saccharose-no acid, no gas,growth Levulose-no acid, no gas, good growth Inosito1--slight alkaline,no gas, good growth Action on milk: Reaction-none; ring growth, orangeOther characteristics:

Nitrites from nitrates Hydrogen su-lfide not produced Indole notproduced No phenol or naphthalene utilized Starch not hydrolyzedUtilizes sodium and amonium salts as nitrogen source The mutantpreviously mentioned and identified as ATCC No. 19,071, like ATCC No.19,070, also has ringsplitting characteristics and produces the muconicacid type of product. This mutant has the same identifying properties astabulated above but, unlike the parent organism, requires a specificvitamin for growth, namely, paminobenzoic acid. In the fermentation ofp-xylene the mutant is more prone to give an accumulation of PTA andDHPT in the broth than is ATCC No. 19,070, although it does notnecessarily produce these products and in many cases will showsubstantial accumulation of DMMA only.

The following examples specifically illustrate embodiments of theinvention. Product analyses for these runs were done by U.V.spectroscopy. Identification of the specific products obtainable hadpreviously been ascertained employing elemental analyses and U.V., LR.and mass spectral procedures.

EXAMPLE I Nocardia corallina ATCC No. 19,070 was used to preparea,u'-DMMA from p-xylene in a 40 1. fermentor operated in continuousmanner as a vortexing system. A mineral salt solution of the approximatecomposition listed above was used and n-hexadecane was employed as thegrowth substrate. The mixture was inoculated with the organism and wasstirred vigorously at about 30 C. while being aerated by suction of airinto the vortex formed by the stirred mixture. First the organism wasallowed to grow in the presence of the hexadecane together with traceamounts of p-xylene until about 5-6 grams of cells had accumulated. Thisrequired about 24 hours. Thereafter the fermentation was continued at 30C. while continuously introducing a mixture of (by volume) 90% p-xyleueand n-hexadecane at a rate such that the p-xylene addition rate was inthe range of 20-40 mL/hr. This maintained the p-xylene concentration inthe fermentation broth in the range of 80-350 ppm. The pH during the runvaried somewhat but generally was within the range of 6.5 to 7.0.Samples of the broth were taken at various times during the fermentationand were analyzed for contents of DMMA, DHPT and also PTA. Nosignificant amount of either of the .latter two compounds was detected.Th DMMA results were:

10 Hours from start: a,oc'-DMMA, g./l. of broth 30 1.8 41 4.5 56 9.8 6511.6

Under the conditions used the DMMA was essentially the only productaccumulated. This shows that the organism ATCC No. 19,070 can be highlyselective in making the muconic acid type product under appropriateconditions.

EXAMPLE II Another run was made under similar conditions as in Example Iexcept that a lower rate of stirring was used and the fermentation wasallowed to proceed for 106 hours. Analysis of the broth at the end ofthat time gave the following results:

a,a'-DMMA g./liter 13.4

PTA g./liter 1.1

DHPT Negligible EXAMPLE III pH: Max. yield of DMMA, g./l. 6.0 4.4 6.58.1 7 O 11.6 8 0 0.06

These results indicate that best production of the muconic acidhomologue is obtained at a pH of the order of 7 and that productformation is markedly decreased at higher pH levels for this partciularmicroorganism. The same is true for ATCC No. 19,070.

EXAMPLE IV Nocardia salmonicolor ATCC No. 19,149 was employed in thefermentation of p-xylene with the objective of producing DHPT. The 40 l.fermentor was used with generally the same procedure as described inExample I. For about the first 36 hours the organism was grown onn-hexadecane at a pH of about 7.0 and thereafter the pH was kept in therange of 7.5-8.0 and a :10 mixture of p-xylene:hexadecane wascontinuously introduced at a rate that maintained the p-xyleneconcentration in the broth at 50-200 ppm. The products in this caseconsisted of DHPT and PTA only and their concentrations in the broth forthree sampling times were as follows:

Cone, g

DHPT PTA Time from Start, hrs:

EXAMPLE V Three runs were made under conditions generally similar tothose used in Example IV and again using ATCC No. 19,149, and thep-xylene concentration in the broth following the growth stage onn-hexadecane was controlled at levels of about 50, and 250 mg./l.,respectively. The initial production rates of DHPT and InitialProduction Yld. at 96 hrs., g./l.

Rate, g./l./hr.

DI-IIT PTA DHPT PTA The data show that increasing the xyleneconcentration within the limits tried suppressed the formation of PTAand increased the maximum yield of DHPT.

EXAMPLE VI This example illustrates the use of cells of Nocardiasalmonicolor ATCC No. 19,149 under non-growth con- 20 dition in thebio-oxidation of p-xylene. First several batches of the cells were grownon n-hexadecane in a 40 l. fermentor at a pH of about 7 for 34 hours.The cells were recovered from the broth by centrifuging and then weresuspended in a phosphate buffer solution containing only 5 Na HPO and KHPO in amounts to maintain pH at about 8. No source materials fornitrogen or trace elements were present. Two batches of the cells inbuffer solution were prepared having cell concentrations respectively ofabout 5 and 15 g./l. A fermentation of each batch at C. was run undervortexing aeration conditions by continuously introducing to thesuspension a 90:10 mixture of p-xylenezn-hexadecane at a rate such thatthe p-xylene concentration in the mixture was maintained at 200-300p.p.m. In each run samples of the broth were taken at times of 16, 26and 34 hours after the addition of p-xylene and were analyzed. Resultswere as follows:

Time from Product c0110., g/l. Addition of p-xylene, hrs. DHPI PTA Cellcone, g./l.:

EXAMPLE VII Shake flask runs were made using three differentdihydroxylating and non-decarboxylating strains of Nocardia in thefermentation of p-xylene. Specifically these were the Nocardiasalmonicolor ATCC No. 19,149 of Examples IV-VI, another strain ofNocardia corallina identified by ATCC No. 19,148 and a Nocardia mim'maidentified by ATCC No. 19,150. The procedure in these runs involvedinoculating 100 ml. of mineral salts solution in a 500 ml. shake flaskwith the organism, adding 0.05 ml. of n-hexadecane, shaking at 30 C. for24 hours, thereafter adding small amounts of p-xylene together withn-hexadecane from time to time and shaking for a total time of the orderof 96 hours. The fermentation beers were then analyzed by U.V.absorptivity. The following tabulation shows the results obtained forduplicate runs with each strain of microorganism.

Product cone, gl../ Species ATCC N0.

DHPT PTA Salmonicolor 19, 149 0.30 1.15 0. 37 1. 08 Corallina 19, 1480.27 1.13 0. 19 1. 07 Miuima 19, 150 0.15 0. 15 0. 15 0. 41

The data show that each of the foregoing strains also can dihydroxylatethe benzene ring without concurrently causing decarboxylation. However,none of these strains seem to cause production of the muconic acidhomologue, at least under the fermentation conditions here employed.

EXAMPLE VIII Two batch runs were made in stirred fermentors usingNocardia cor-allina designated as ATCC No. 19,070 and ATCC No. 19,071,respectively. In each run a mixture of 480 ml. of an anion exchangeresin and sufficient mineral salts solution to make a total volume of3000 ml. were used. The medium also contained 0.2% peptone and 0.1% beefextract and its pH was maintained at about 6.5. Following inoculationthe organisms were allowed to grow on n-hexadecane for 36 hours andthereafter p-xylene was added in small amounts from time to time whilethe mixture was being stirred and aerated at 30 C. Each run wasconducted for total hours and 24 ml. of p-xylene total were used. Thekinds and amounts of products, including those absorbed on the anionexchange resin as well as those in the broth, were then determined, thecombined results being as follows:

Product amount, g./l. of broth Product ATCC No. 19,070 ATCC No. 19,071

DMMA 12. 2 14. 6 O. 7 0. 8 1. 0 0. 8 0. 5 0. 6

3,6-dimethyleatech0l.

From these data it can be seen that both microorganisms, under theconditions used in these runs, can cause accumulation of both thesubstituted muconic acid product and the substituted2,3-dihydroxybenzoic acid product. Further, the data also show thatunder some circumstances it is possible for the substituted catecholinter mediate to accumulate, although the proportion thereof is minorfor the specific strains of microorganisms here used.

In the last-described example an anion exchange resin was, as stated,employed in the fermentation mixture during the fermentation. The use ofsuch resin during fermentation causes an unexpected improvement in theyield of desired acid product. However, such use of resin is not claimedherein, as this constitutes the subject matter of the copending Humphreyand Raymond application Ser. No. 512,543, filed Dec. 8, 1965.

The foregoing examples illustrate the preparation of methyl-substitutedmuconic acids and/or 2,3-dihydroxybenzoic acids from C -C methylbenzenesas herein specified. While the examples are specifically directed to thebio-oxidation of p-xylene, fermentations with other methylbenzenes asherein specified give analogous results. For these other substratehydrocarbons the fermentation reactions proceed by analogous metabolicpaths, so that one or the other or both of these kinds of acids areproduced whenever dihydroxylating and non-decarboxylating strains ofNocardia are employed to effect the fermentation.

The acids that can be prepared by the present invention are valuableproducts having various applications of commercial interest. Forexample, the substituted muconic acids, being di-terminal acids, areuseful in the preparation of polymers of various types. Thedihydroxybenzoic acids have utility as chelating agents, metaldeactivators and dye intermediates.

We claim:

1. Method of producing organic acid having 7-10 carbon atoms, said acidbeing a methyl-substituted muconic acid or a 2,3-dihydroxybenzoic acidor both, which comprises subjecting a C -C methylbenzene having 1-4methyl groups and at least two consecutive unsubstituted ring carbonatoms in the presence of a nutrient medium and under fermentationconditions to the action of an orthodihydroxylating andnondecarboxylating strain of Nocardia and recovering an acid of at leastone of the aforesaid acid types from the fermentation mixture.

2. Method according to claim 1 wherein said fermentation conditionsinclude a pH level above 7 and a 2,3- dihydroxybenzoic acid isrecovered.

3. Method according to claim 1 wherein said fermentation conditionsinclude a pH level below 7 and a methylsubstituted muconic acid isrecovered.

4. Method according to claim 1 wherein said strain is a member of thespecies Nocardia corallina, Nocardia salmonicolor or Nocardia minima.

5. Method according to claim 1 wherein said strain is ATCC No. 19,070,ATCC No. 19,071, ATCC No. 19,148, ATCC No. 19,149 or ATCC No. 19,150.

6. Method according to claim 1 wherein said methylbenzene is p-xylene.

7. Method according to claim 6 wherein said strain is a member of thespecies Nocardia corallz'na, Nocardia salmonicolor or Nocardia minima.

8. Method according to claim 7 wherein 2,3-dihydroxyp-toluic acid isrecovered.

9. Method according to claim 7 wherein a,a'-dimethylmuconic acid isrecovered.

10. Method according to claim 7 wherein both 2,3-dihydroxy-p-toluic acidand a,a'-dimethylmuconic acid are recovered.

11. Method according to claim 6 wherein said strain is Nocardiacorallina ATCC No. 19,070 or ATCC No. 19,071.

12. Method according to claim 1 wherein said methylbenzene is p-xylene,said strain is a member of the species Nocardia corallina, saidfermentation conditions include a pH below 7 and u,a'-dimethylmuconicacid is recovered.

13. Method according to claim 1 wherein said methylbenzene is p-xylene,said strain is a member of the species Nocardia salmonicolor, saidfermentation conditions include a pH above 7 and 2,3-dihydroxy-p-toluicacid is recovered.

14. Method according to claim 1 wherein said methylbenzene is a xyleneand a monomethyl-Z,3-dihydroxybenzoic acid, a dimethylmuconic' acid orboth are recovered from the fermentation mixture.

15. Method according to claim 14 wherein said strain is a member of thespecies Nocara'ia corallina, Nocardia salmonicolor or Nocardia minima.

16. Method according to claim 14 wherein said strain is ATCC No. 19,070,ATCC No. 19,071, ATCC No. 19,148, ATCC No. 19,149 or ATCC No. 19,150.

17. Method of preparing a,a'-dimethylmuconic acid which comprisessubjecting p-xylene in the presence of a nutrient medium and underfermentation conditions including a pH level not substantially above 7to the action of an orthodihydroxylating, non-decarboxylating andring-splitting strain of Nocardia and recovering said acid from thefermentation broth.

18. Method according to claim 17 wherein said strain is of the speciesNocardza corallina and said conditions include a. pH in the approximaterange of 6-7.

19. Method according to claim 18 wherein said strain is ATCC No. 19,070or ATCC No. 19,071.

20. Method according to claim 1 wherein said strain is ATCC No. 19,070or ATCC No. 19,071.

References Cited UNITED STATES PATENTS LIONEL M. SHAPIRO, PrimaryExaminer.

